The invention relates to methods and kits for increasing a ratio of rare cells to non-rare cells in a blood sample and for identifying the rare cells. More particularly, the invention relates to methods and kits for increasing a ratio of cancer cells to white blood cells in a blood sample and for identifying the cancer cells.
Cell filtration for the separation of cancer cells using a porous matrix is used to sort cells by size and, in most instances, such filtration methods allow for the extraction of cells following separation. Both microfluidic post and microfluidic membrane methods are used in these filtration approaches. However, the existing filtration methods are limited by certain factors, which include, for example, the range of diameters that in vitro cells have rather than a single diameter. This range of diameters is demonstrated, for example, in the case of cancer cell populations and white blood cell populations, which have overlapping diameters. During filtration small cancer cells are lost and larger white blood cells contaminate the separated material. Furthermore, cancer cell populations and white blood cell populations are very heterogeneous and comprise a variety of cell diameter types within these individual populations. For example, the range of diameters for white blood cells is much wider when considering samples including populations of neutrophils, eosinophils, basophils, macrophages, lymphocytes and macrophages. Cancer cells in blood can also range in size.
Another limitation on the selectivity of a cell filtration method is that ideal pore size is impacted by the deformability of various cells. This deformability further reduces selectivity for size exclusion to isolate different cells. A small diameter cell with low deformability requires the use of a matrix having even a smaller pore size than otherwise might be used. Using smaller pore sizes increases the number of desired cells captured (e.g., cancer cells) but results in a less pure separation. Furthermore, when the pores are made smaller, they are prone to clogging. Pressure increases as pore size decreases. Clogging and higher pressure can be temporarily reduced by an oscillatory flow force (on/off). Preventing a pressure from building across the separation microstructure is important as higher pressures reduce the impact of cell size on separation.
In addition, blood cells are typically fixed before separation by filtration to improve separation of the cells. Recovery of target cells is reduced when fixation is not used since some target cells have higher deformability than other target cells. Fixation causes all cells to have similar deformability (viscoelastic properties). However, there are a number of disadvantages to fixing cells such as, for example, the requirement of greater pressure for passage of the fixed cells through a porous matrix. As pressure increases, ideal pore size decreases. Smaller pores lead to greater capture of undesired cells. Another disadvantage is that fixed cells are not viable and cannot be grown or used to measure cells responses to stimulus.
There is, therefore, a need to develop a filtration separation method that does not require fixation. The method should improve cell recovery and be independent of differences in cell diameter and differences in viscoelastic properties.